Flagellin gene, flaC of Campylobacter

ABSTRACT

Purified and isolated nucleic acid molecules are provided which encode a FlaC flagellin protein of a strain of Campylobacter, particularly  C. jejuni,  or a fragment or an analog of the FlaC flagellin protein. The nucleic acid molecules may be used to produce proteins free of contaminants derived from bacteria normally containing the FlaA or FlaB proteins for purposes of diagnostics and medical treatment. Furthermore, the nucleic acid molecules, proteins encoded thereby and antibodies raised against the proteins, may be used in the diagnosis of infection.

FIELD OF THE INVENTION

[0001] The present invention is related to the molecular cloning of agene encoding a flagellin protein, identified herein as FlaC, of theflagellar filament from a strain of Campylobacter.

BACKGROUND OF THE INVENTION

[0002]Campylobacter jejuni is a Gram-negative spiral microaerophilicbacterium that has been recognized as a cause of secretory type diarrheaand enteritis (Ref. 1). Throughout this application, various referencesare referred to in parenthesis to more fully describe the state of theart to which this invention pertains. Full bibliographic information foreach, citation is found at the disclosed end of the specificationimmediately preceding the claims. These references are herebyincorporated by reference into the present disclosure). The flagellum ofC. jejuni is responsible for bacterial motility which enhances theorganism's pathogenicity. The flagellum consists of three majorcomponents; the filament, the hook, and the basal body (Ref. 2). Acampylobacter cell carries a single unsheathed flagellum at one or bothpoles of the body. The flagella are responsible for the high motility ofthe organisms as aflagellate mutants are nonmotile (Refs. 3, 4, 5, 6, 7,8, 9). A number of studies indicated that the polar flagellum plays animportant role in colonization of the viscous mucus lining of thegastric intestinal tract and that it is an important virulencedeterminant (Refs. 3,4,7,10,11).

[0003] The basic structure of the bacterial flagellum consists of apropeller (filament) connected via a universal joint (hook) to atransmission shaft, motor and brushings (basal body) embedded in thecell envelope (Ref. 12). The flagellar filament consists of severalthousand self-assembling protein (flagellin) monomers arranged in ahelix. These form a hollow tube of relatively constant diameter andvariable length with an over corkscrew morphology.

[0004] Most eubacterial flagellar filaments that have been characterizedappear to be composed of a single kind of flagellin (Ref. 8). However anumber of Eubacteria have now been shown to possess multipleflagellingenes (Refs. 6, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22), Cjejuni (Refs. 13, 15, 17, 21) and C. coli (Refs. 6, 13) have beenreported to have two flagellin genes (flaA and flaB). In C. jejuni, theflagellin genes flaA and flaB have been isolated and sequenced, howeverprior to the present invention a third flagellin gene had not beenisolated and characterized.

[0005] It would be advantageous to provide nucleic acid moleculesencoding flagellin proteins of the flagella for strains of Campylobacterand purified flagellin proteins, including flaC for use as antigens,immunogenic compositions, including vaccines, carriers for otherantigens and immunogens and the generation of diagnostic reagents.

SUMMARY OF THE INVENTION

[0006] The present invention is directed towards the provision ofpurified and isolated nucleic acid molecules encoding a flagellinprotein C (FlaC) of a strain of Campylobacter or a fragment or an analogof the flagellin protein. The nucleic acid molecules provided herein areuseful for the specific detection of strains of Campylobacter, and fordiagnosis of infection by Campylobacter. The purified and isolatednucleic acid molecules provided herein, such as DNA, are also useful forexpressing the flaC gene by recombinant DNA means for providing, in aneconomical manner, purified and isolated FlaC proteins, subunits,fragments or analogs thereof. The FlaC protein, subunits or fragmentsthereof or analogs thereof, as well as nucleic acid molecules encodingthe same and vectors containing such nucleic acid molecules, are usefulin immunogenic compositions against diseases caused by Campylobacter,the diagnosis of infection by Campylobacter and as tools for thegeneration of immunological reagents. Monoclonal antibodies ormono-specific antisera (antibodies) raised against the FlaC proteinproduced in accordance with aspects of the present invention are usefulfor the diagnosis of infection by Campylobacter, the specific detectionof Campylobacter (in for example in vitro and in vivo assays) and forthe treatment of diseases caused by Campylobacter.

[0007] Peptides corresponding to portions of the FlaC protein or analogsthereof are useful immunogenic compositions against disease caused byCampylobacter, the diagnosis of infection by Campylobacter and as toolsfor the generation of immunological reagents. Monoclonal antibodies orantisera raised against these peptides, produced in accordance withaspects of the present invention, are useful for the diagnosis ofinfection by Campylobacter, the specific detection of Campylobacter (in,for example, in vitro and in vivo assays) and for use in passiveimmunization as a treatment of disease caused by Campylobacter.

[0008] In accordance with one aspect of the present invention, there isprovided a purified and isolated nucleic acid molecule encoding aflagellin protein (FlaC) of flagellum of a strain of Campylobacter, moreparticularly, a strain of Campylobacter jejunis, or a fragment or ananalog of the FlaC protein.

[0009] In one preferred embodiment of the invention, the nucleic acidmolecule may encode the FlaC protein of the Campylobacter strain.

[0010] In another aspect of the present invention, there is provided apurified and isolated nucleic acid molecule having a nucleotide sequenceselected from the group consisting of: (a) the entire nucleotidesequence set out in FIG. 1 (SEQ ID No: 1), or the complementary sequenceof said sequence; (b) the coding nucleotide sequence set out in FIG. 1(SEQ ID No: 2), or the complementary sequence of said sequence; (c) anucleotide sequence encoding the amino acid sequence set forth in FIG. 1(SEQ ID No: 3); and (d) a nucleotide sequence which hybridizes understringent conditions to any one of the sequences defined in (a), (b) or(c). The DNA sequence defined in (c) preferably has at least about 90%sequence identity with any one of the DNA sequences defined in (a) and(b).

[0011] In an additional aspect, the present invention includes a vectoradapted for transformation of a host, comprising a nucleic acid moleculeas provided herein. The vector may be one having the characteristics ofplasmid pD2-2.

[0012] The plasmids may be adapted for expression of the encoded FlaCprotein, fragments or analogs thereof, in a heterologous or homologoushost, in either a lipidated or non-lipidated form. Accordingly, afurther aspect of the present invention provides an expression vectoradapted for transformation of a host comprising a nucleic acid moleculeas provided herein and expression means operatively coupled to thenucleic acid molecule for expression by the host of the FlaC protein orthe fragment or analog of the FlaC protein. In specific embodiments ofthis aspect of the invention, the nucleic acid molecule may encodesubstantially all the FlaC protein of the Campylobacter strain. Theexpression means may include a nucleic acid portion encoding a leadersequence for secretion from the host of the FlaC protein or the fragmentor the analog of the FlaC protein. The expression means also may includea nucleic acid portion encoding a lipidation signal for expression fromthe host of a lipidated form of the FlaC protein or the fragment or theanalog of the FlaC protein. The host may be selected from, for example,Escherichia coli, Bordetella, Bacillus, Haemophilus, Moraxella, fungi,yeast or baculovirus and Semliki Forest virus expression systems may beused.

[0013] In an additional aspect of the invention, there is provided atransformed host containing an expression vector as provided herein. Theinvention further includes a recombinant FlaC protein or fragment oranalog thereof producible by the transformed host. Further aspects ofthe present invention provide an isolated and purified FlaC protein of aCampylobacter strain substantially free from other proteins of theCampylobacter strain. The Campylobacter strain may be C. jejuni.

[0014] The present invention further provides synthetic peptidescorresponding to portions of the FlaC protein. Accordingly, in a furtheraspect of the invention, there is provided a synthetic peptide having noless than six amino acids and no more than 150 amino acids andcontaining an amino acid sequence corresponding to a portion only of aFlaC protein of a strain of Campylobacter or of a fragment or an analogof the FlaC protein.

[0015] In accordance with another aspect of the invention, animmunogenic composition is provided which comprises at least one activecomponent selected from at least one nucleic acid molecule as providedherein, at least one recombinant protein as provided herein, at leastone of the purified and isolated FlaC protein, as provided herein and atleast one synthetic peptide as provided herein, and a pharmaceuticallyacceptable carrier therefor or vector therefor. The at least one activecomponent produces an immune response when administered to a host.

[0016] The immunogenic compositions provided herein may be formulated asa vaccine for in vivo administration to protect against diseases causedby bacterial pathogens that produce flagellin proteins. For suchpurpose, the compositions may be formulated as a microparticle, capsule,ISCOM or liposome preparation. Alternatively, the compositions may beprovided in combination with a targeting molecule for delivery tospecific cells of the immune system or to mucosal surfaces. Theimmunogenic composition may comprise a plurality of active components toprovide protection against disease caused by a plurality of species offlagellin protein producing bacteria.

[0017] The immunogenic compositions of the invention (includingvaccines) may further comprise at least one other immunogenic orimmunostimulating material and the immunostimulating material may be atleast one adjuvant or at least one cytokine. Suitable adjuvants for usein the present invention include (but are not limited to) aluminumphosphate, aluminum hydroxide, QS21, Quil A, derivatives and componentsthereof, ISCOM matrix, calcium phosphate, calcium hydroxide, zinchydroxide, a glycolipid analog, an octadecyl ester of an amino acid, amuramyl dipeptide polyphosphazene, ISCOPREP, DC-chol, DDBA and alipoprotein. Advantageous combinations of adjuvants are described incopending U.S. patent application Ser. Nos. 08/261,194 filed Jun. 16,1994 and 08/483,856, filed Jun. 7, 1995, assigned to the assignee hereofand the disclosures of which are incorporated herein by referencethereto.

[0018] In accordance with another aspect of the invention, there isprovided a method for inducing protection against infection or diseasecaused by Campylobacter or other bacteria that produce flagellinprotein, comprising the step of administering to a susceptible host,which may be a primate, such as a human, an effective amount of theimmunogenic composition as recited above.

[0019] In accordance with another aspect of the invention, an antiserumor antibody specific for the recombinant protein, the isolated andpurified FlaC protein, synthetic peptide or the immunogenic composition,is provided.

[0020] In a further aspect, there is provided a live vector for deliveryof FlaC protein to a host, comprising a vector containing the nucleicacid molecule as described above. The vector may be selected fromSalmonella, BCG, adenovirus, poxvirus, vaccinia and poliovirus. Thenucleic acid molecule may encode a fragment of the FlaC protein of aCampylobacter strain which is conserved among bacteria that produce theFlaC protein. Such vector may be included in an immunogenic compositionprovided herein.

[0021] The present invention further includes a method of determiningthe presence of nucleic acid encoding the FlaC protein of a strain ofCampylobacter, in a sample, comprising the steps of: (a) contacting thesample with the nucleic acid molecule provided herein to produceduplexes comprising the nucleic acid molecule and any said nucleic acidmolecule encoding the FlaC protein of Campylobacter present in thesample and specifically hybridizable therewith; and (b) determiningproduction of the duplexes.

[0022] In an additional aspect, the present invention provides a methodof determining the presence of a FlaC protein of a Campylobacter strainin a sample, comprising the steps of (a) immunizing a subject with theimmunogenic composition provided herein to produce antibodies specificfor the FlaC protein; (b) contacting the sample with the antibodies toproduce complexes comprising FlaC protein of a Campylobacter strainpresent in the sample and the FlaC protein specific antibodies; anddetermining production of the complexes.

[0023] A further aspect of the present invention provides a diagnostickit for determining the presence of nucleic acid encoding the FlaCprotein of a strain of Campylobacter, in a sample, comprising (a) thenucleic acid molecule provided herein; (b) means for contacting thenucleic acid with the sample to produce duplexes comprising the nucleicacid molecule and any said nucleic acid present in the sample andhybridizable with the nucleic acid molecule; and (c) means fordetermining production of the duplexes.

[0024] In another aspect of the present invention, there is provided adiagnostic kit for detecting the presence of a FlaC protein of aCampylobacter strain in a sample, comprising (a) a FlaC protein specificantibody to the immunogenic composition provided herein; (b) means forcontacting the antibody with the sample to produce a complex comprisingsaid FlaC protein and the antibody; and (c) means for determiningproduction of the complex.

[0025] The invention further includes the use of the nucleic acidmolecules and proteins provided herein as medicines. The inventionadditionally includes the use of the nucleic acid molecules and proteinsprovided herein in the manufacture of medicaments for protection againstinfection by strains of Campylobacter.

[0026] The purified and isolated DNA molecules comprising at least aportion coding for a FlaC protein of a species of Campylobacter typifiedby the embodiments described herein are advantageous as:

[0027] nucleic acid probes for the specific identification ofCampylobacter strains in vitro or in vivo.

[0028] the products encoded by the DNA molecules are useful asdiagnostic reagents, antigens for the production ofCampylobacter-specific antisera, for vaccination against the diseasescaused by species of Campylobacter and (for example) detecting infectionby Campylobacter.

[0029] peptides corresponding to portions of the FlaC protein astypified by the embodiments described herein are advantageous asdiagnostic reagents, antigens for the production ofCampylobacter-specific antisera, for vaccination against the diseasescaused by species of Campylobacter and (for example) for detectinginfection by Campylobacter.

BRIEF DESCRIPTION OF DRAWINGS

[0030] The present invention will be further understood from thefollowing description with reference to the drawings in which:

[0031]FIG. 1 shows the nucleotide sequence of flaC gene and flankingregions (SEQ ID No: 1). The translated amino acid (SEQ ID No: 3) islocated below the encoding sequence (SEQ ID No: 2). The ribosome bindingsite (RBS), the −10 and −35 regions are indicated by lines above thesequence. The putative transcriptional start sites are indicated by anasterisk below the sequence. Inverted repeats are shown with an arrowbelow the sequence.

[0032]FIG. 2 contains the restriction map of clone pD2-2 in pBluescriptvector. The location of the flaC is denoted by the boxed area below therestriction map. The restriction sites are: B, BglII; C, ClaI; H,HindIII; P, PvuI; R, RsaI; S, SalI; and X, XbaI. The shaded boxrepresents vector sequences. The direction of transcription is indicatedby the arrow.

[0033]FIG. 3 shows Southern Blot hybridization with flaC to HindIIIdigested genomic DNA from various Campylobacter species using 30%formamide. Lane 1, 100 bp molecular marker; lane 2, blank; lane 3, C.jejuni ATCC 43431; lane 4, C. jejuni OH4382; lane 5, blank; lane 6, C.jejuni ATCC 43446; lane 7, C. jejuni ATCC 33559; lane 8, C. coli ATCC43474; lane 9, C. lari ATCC 35221; lane 10, C. lari PC637; Lane 11, C.upsaliensis ATCC 43954; lane 12, blank; lane 13, 100 bp molecularmarker.

GENERAL DESCRIPTION OF THE INVENTION

[0034] Any Campylobacter strain may be conveniently used to provide thepurified and isolated nucleic acid, provided herein which may be in theform of DNA molecules, comprising at least a portion of the nucleic acidcoding for FlaC protein of a flagellum as typified by embodiments of thepresent invention. Such strains are generally available from clinicalsources and from bacterial culture collections, such as the AmericanType Culture Collection, Rockville, Md., U.S.A. One particular usefulspecies is C. jejuni.

[0035] In this application, the term “a flagellin protein C” is used todefine a family of FlaC proteins which includes those having variationsin their amino acid sequences including those naturally occurring invarious strains of Campylobacter. The purified and isolated DNAmolecules comprising at least a portion coding for the FlaC protein ofthe present invention also include those encoding fragments orfunctional analogs of the FlaC protein. In this application, a firstprotein is a “functional analog” of a second protein if the firstprotein is immunologically related to and/or has the same function asthe second protein. The functional analog may be, for example, afragment of the protein or a substitution, addition or deletion mutantthereof.

[0036] Oligonucleotide probes derived from the Fur-box sequences fromthe recently cloned C. jejuni fur gene (Ref. 4) were used to screen apBluescript genomic library of C. jejuni. One of the clones sequenced,pD2-2 had an open reading frame (ORF) that codes for a polypeptide of249 amino acid residues. This protein had the highest homology with theN-terminal region of the C. coli FlaA protein sequence. The open readingframe was designated FlaC as the third flagellin gene in Campylobacter.

[0037] It is clearly apparent to one skilled in the art, that thevarious embodiments of the present invention have many applications inthe fields of vaccination, diagnosis, treatment of, for example,Campylobacter infections, and infections with other bacterial pathogensthat produce FlaC protein and the generation of immunological reagents.A further non-limiting discussion of such uses is further presentedbelow.

[0038] 1. Vaccine Preparation and Use

[0039] Immunogenic compositions, suitable to be used as vaccines, may beprepared from FlaC proteins, analogs and fragments thereof, peptides andnucleic acid molecules encoding such FlaC proteins, fragments andanalogs thereof and peptides as disclosed herein. The vaccine elicits animmune response which produces antibodies, including anti-FlaC proteinantibodies and antibodies that are opsonizing or bactericidal. Shouldthe vaccinated subject be challenged by Campylobacter or other bacteriathat produce a FlaC protein, the antibodies bind to the basal body rodprotein and thereby inactivate the bacteria. Opsonizing or bactericidalantibodies may be particularly useful for providing protection.

[0040] Vaccines containing peptides are generally well known in the art,as exemplified by U.S. Pat. Nos. 4,601,903; 4,599,231; 4,599,230; and4,596,792; all of which references are incorporated herein by reference.Immunogenic compositions including vaccines may be prepared asinjectables, as liquid solutions or emulsions. The nucleic acidmolecules, FlaC protein, analogs and fragments thereof and/or peptidesmay be mixed with pharmaceutically acceptable excipients which arecompatible with the FlaC protein, fragments analogs or peptides. Suchexcipients may include, water, saline, dextrose, glycerol, ethanol, andcombinations thereof. The immunogenic compositions and vaccines mayfurther contain auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, or adjuvants to enhance the effectivenessof the vaccines. Immunogenic compositions and vaccines may beadministered parenterally, by injection subcutaneously orintramuscularly. Alternatively, the immunogenic compositions formedaccording to the present invention, may be formulated and delivered in amanner to evoke an immune response at mucosal surfaces. Thus, theimmunogenic composition may be administered to mucosal surfaces by, forexample, the nasal or oral (intragastric) routes. The immunogeniccomposition may be provided in combination with a targeting molecule fordelivery to specific cells of the immune system or to mucosal surfaces.Some such targeting molecules include vitamin B12 and fragments ofbacterial toxins, as described in WO 92/17167 (Biotech Australia Pty.Ltd.), and monoclonal antibodies, as described in U.S. Pat. No.5,194,254 (Barber et al). Alternatively, other modes of administrationincluding suppositories and oral formulations may be desirable. Forsuppositories, binders and carriers may include, for example,polyalkalene glycols or triglycerides. Oral formulations may includenormally employed incipients such as, for example, pharmaceutical gradesof saccharine, cellulose and magnesium carbonate. These compositionstake the form of solutions, suspensions, tablets, pills, capsules,sustained release formulations or powders and contain 1 to 95% of thenucleic acid molecule, FlaC protein, fragment analogs and/or peptides.

[0041] The vaccines are administered in a manner compatible with thedosage formulation, and in such amount as will be therapeuticallyeffective, protective and immunogenic. The quantity to be administereddepends on the subject to be treated, including, for example, thecapacity of the individual's immune system to synthesize antibodies, andif needed, to produce a cell-mediated immune response. Precise amountsof active ingredient required to be administered depend on the judgmentof the practitioner. However, suitable dosage ranges are readilydeterminable by one skilled in the art and may be of the order ofmicrograms of the FlaC protein, analogs and fragments thereof and/orpeptides. Suitable regimes for initial administration and booster dosesare also variable, but may include an initial administration followed bysubsequent administrations. The dosage of the vaccine may also depend onthe route of administration and will vary according to the size of thehost.

[0042] Thus, the nucleic acid molecules encoding the FlaC protein,fragments or analogs thereof, of the present invention may also be useddirectly for immunization by administration of the nucleic acid molecule(including DNA molecules) directly, for example by injection for geneticimmunization or by constructing a live vector such as Salmonella, BCG,adenovirus, poxvirus, vaccinia or poliovirus. A discussion of some livevectors that have been used to carry heterologous antigens to the immunesystem are discussed in, for example, O'Hagan (Ref. 23). Processes forthe direct injection of DNA into test subjects for genetic immunizationare described in, for example, Ulmer et al. (Ref. 24).

[0043] The use of peptides in vivo may first require their chemicalmodification since the peptides themselves may not have a sufficientlylong serum and/or tissue half-life and/or sufficient immunogenicity.Such chemically modified peptides are referred to herein as “peptideanalogs”. The term “peptide analog” extends to any functional chemicalequivalent of a peptide characterized by its increased stability and/orefficacy and immunogenicity in vivo or in vitro in respect of thepractice of the invention. The term “peptide analog” is also used hereinto extend to any amino acid derivative of the peptides as describedherein. Peptide analogs contemplated herein are produced by proceduresthat include, but are not limited to, modifications to side chains,incorporation of unnatural amino acids and/or their derivatives duringpeptide synthesis and the use of cross-linkers and other methods whichimpose conformational constraint on the peptides or their analogs.

[0044] Examples of side chain modifications contemplated by the presentinvention include modification of amino groups such as by reductivealkylation by reaction with an aldehyde followed by reduction withNaBH₄; amidation with methylacetimidate; acetylation with aceticanhydride; carbamylation of amino groups with cyanate;trinitrobenzylation of amino groups with 2, 4, 6, trinitrobenzenesulfonic acid (TNBS); alkylation of amino groups with succinic anhydrideand tetrahydrophthalic anhydride; and pyridoxylation of lysine withpyridoxal-5′-phosphate followed by reduction with NaBH₄.

[0045] The guanidino group of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

[0046] The carboxyl group may be modified by carbodimide activation viao-acylisourea formation followed by subsequent derivatisation, forexample, to a corresponding amide.

[0047] Sulfhydryl groups may be modified by methods such ascarboxymethylation with iodoacetic acid or iodoacetamide; performic acidoxidation to cysteic acid; formation of mixed disulphides with otherthiol compounds; reaction with maleimide; maleic anhydride or othersubstituted maleimide; formation of mercurial derivatives using4-chloromercuribenzoate, 4-chloromercuriphenylsulfonic acid,phenylmercury chloride, 2-chloromercuric-4-nitrophenol and othermercurials; carbamylation with cyanate at alkaline pH.

[0048] Tryptophan residues may be modified by, for example, oxidationwith N-bromosuccinimide or alkylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphonyl halides. Tryosine residuesmay be altered by nitration with tetranitromethane to form a3-nitrotyrosine derivative.

[0049] Modification of the imidazole ring of a histidine residue may beaccomplished by alkylation with iodoacetic acid derivatives orN-carbethoxylation with diethylpyrocarbonate.

[0050] Examples of incorporating unnatural amino acids and derivativesduring peptide synthesis include, but are not limited to, use ofnorleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoicacid, 6-aminohexanoic acid-, t-butylglycine, norvaline, phenylglycine,ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid,2-thienyl alanine and/or D-isomers of amino acids.

[0051] Immunogenicity can be significantly improved if the antigens areco-administered with adjuvants, commonly used as an 0.05 to 1.0 percentsolution in phosphate—buffered saline. Adjuvants enhance theimmunogenicity of an antigen but are not necessarily immunogenicthemselves. Adjuvants may act by retaining the antigen locally near thesite of administration to produce a depot effect facilitating a slow,sustained release of antigen to cells of the immune system. Adjuvantscan also attract cells of the immune system to an antigen depot andstimulate such cells to elicit immune responses.

[0052] Immunostimulatory agents or adjuvants have been used for manyyears to improve the host immune responses to, for example, vaccines.Intrinsic adjuvants, such as lipopolysaccharides, normally are thecomponents of the killed or attenuated bacteria used as vaccines.Extrinsic adjuvants are immunomodulators which are typicallynon-covalently linked to antigens and are formulated to enhance the hostimmune responses. Thus, adjuvants have been identified that enhance theimmune response to antigens delivered parenterally. Some of theseadjuvants are toxic, however, and can cause undesirable side-effects,making them unsuitable for use in humans and many animals. Indeed, onlyaluminum hydroxide and aluminim phosphate (collectively commonlyreferred to as alum) are routinely used as adjuvants in human andveterinary vaccines. The efficacy of alum in increasing antibodyresponses to diptheria and tetanus toxoids is will established and aHBsAg vaccine has been adjuvanted with alum. While the usefulness ofalum is well established for some applications, it has limitations. Forexample, alum is ineffective for influenza vaccination andinconsistently elicits a cell mediated immune response. The antibodieselicited by alum-adjuvanted antigens are mainly of the IgG1 isotype inthe mouse, which may not be optimal for protection by some vaccinalagents.

[0053] A wide range of extrinsic adjuvants can provoke potent immuneresponses to antigens. These include saponins complexed to membraneprotein antigens (immune stimulating complexes ISCOMs), pluronicpolymers with mineral oil, killed mycobacteria and mineral oil, Freund'scomplete adjuvant, bacterial products, such as muramyl dipeptide (MDP)and lipopolysaccharide (LPS), as well as lipid A, and liposomes.

[0054] To efficiently induce humoral immune responses (HIR) andcell-mediated immunity (CMI), immunogens are emulsified in adjuvants.Many adjuvants are toxic, inducing granulomas, acute and chronicinflammations (Freund's complete adjuvant, FCA), cytolysis (saponins andpluronic polymers) and pyrogenicity, arthritis and anterior uveitis (LPSand MDP). Although FCA is an excellent adjuvant and widely used inresearch, it is not licensed for use in human or veterinary vaccinesbecause of its toxicity.

[0055] Desirable characteristics of ideal adjuvants include:

[0056] (1) lack of toxicity;

[0057] (2) ability to stimulate a long-lasting immune response;

[0058] (3) simplicity of manufacture and stability in long-term storage;

[0059] (4) ability to elicit both CMI and HIR to antigens administeredby various routes, if required;

[0060] (5) synergy with other adjuvants;

[0061] (6) capability of selectively interacting with populations ofantigen presenting cells (APC);

[0062] (7) ability to specifically elicit appropriate T_(H)1 or T_(H)2cell-specific immune responses; and

[0063] (8) ability to selectively increase appropriate antibody isotypelevels (for example, IgA) against antigens.

[0064] U.S. Pat. No. 4,855,283 granted to Lockhoff et al on Aug. 8, 1989which is incorporated herein by reference thereto teaches glycolipidanalogues including N-glycosylamides, N-glycosylureas andN-glycosylcarbamates, each of which is substituted in the sugar residueby an amino acid, as immuno-modulators or adjuvants. Thus, Lockhoff etal. 1991 reported that N-glycolipid analogs displaying structuralsimilarities to the naturally-occurring glycolipids, such asglycosphingolipids and glycoglycerolipids, are capable of elicitingstrong immune responses in both herpes simplex virus vaccine andpseudorabies virus vaccine. Some glycolipids have been synthesized fromlong chain-alkylamines and fatty acids that are linked directly with thesugars through the anomeric carbon atom, to mimic the functions of thenaturally occurring lipid residues.

[0065] U.S. Pat. No. 4,258,029 granted to Moloney and incorporatedherein by reference thereto, teaches that octadecyl tyrosinehydrochloride (OTH) functions as an adjuvant when complexed with tetanustoxoid and formalin inactivated type I, II and III poliomyelitis virusvaccine. Also, Nixon-George et al. (Ref. 25), reported that octadecylesters of aromatic amino acids complexed with a recombinant hepatitis Bsurface antigen, enhanced the host immune responses against hepatitis Bvirus.

[0066] Lipidation of synthetic peptides has also been used to increasetheir immunogenicity. Thus, Weismuller (Ref. 26), describes a peptidewith a sequence homologous to a foot-and-mouth disease viral proteincoupled to an adjuvant tripalmityl-s-glyceryl-cysteinylserylserine,being a synthetic analogue of the N-terminal part of the lipoproteinfrom Gram negative bacteria. Furthermore, Deres et al. (Ref. 27),reported in vivo priming of virus-specific cytotoxic T-lymphocytes withsynthetic lipopeptide vaccine which comprised of modified syntheticpeptides derived from influenza virus nucleoprotein by linkage to alipopeptide, N-palmityl-s-[2,3-bis(palmitylxy)-(2RS)-propyl-[R]-cysteine(TPC).

[0067] 2. Immunoassays

[0068] The FlaC protein, analogs and fragments thereof and/or peptidesof the present invention are useful as immunogens, as antigens inimmunoassays including enzyme-linked immunosorbent assays (ELISA), RIAsand other non-enzyme linked antibody binding assays or procedures knownin the art for the detection of anti-bacterial, Campylobacter, FlaCprotein and/or peptide antibodies. In ELISA assays, the FlaC proteins,analogs, fragments and/or peptides corresponding to portions of FlaCprotein are immobilized onto a selected surface, for example a surfacecapable of binding proteins or peptides such as the wells of apolystyrene microtiter plate. After washing to remove incompletelyadsorbed FlaC protein, analogs, fragments and/or peptides, a nonspecificprotein such as a solution of bovine serum albumin (BSA) or casein thatis known to be antigenically neutral with regard to the test sample maybe bound to the selected surface. This allows for blocking ofnonspecific adsorption sites on the immobilizing surface and thusreduces the background caused by nonspecific bindings of antisera ontothe surface. The selected peptides may be from the conserved regions ofFlaC protein to enhance the cross-species detection unless oneparticular bacterial species is to be detected. In that event, apolypeptide is selected which is unique to the FlaC protein of thatparticular species. Normally, the peptides are in the range of 12residues and up and preferably 14 to 30 residues. It is understoodhowever, that a mixture of peptides may be used either as an immunogenin a vaccine or as a diagnostic agent. There may be circumstances wherea mixture of peptides from the conserved regions and/or from thenon-conserved regions are used to provide cross-species protectionand/or specific diagnosis. In this instance, the mixture of peptideimmunogens is commonly referred to as a “cocktail” preparation for useas a vaccine or diagnostic agent.

[0069] The immobilizing surface is then contacted with a sample such asclinical or biological materials to be tested in a manner conducive toimmune complex (antigen/antibody) formation. This may include dilutingthe sample with diluents such as BSA, bovine gamma globulin (BGG) and/orphosphate buffered saline (PBS)/Tween. The sample is then allowed toincubate for from 2 to 4 hours, at temperatures such as of the order of25° to 37° C. Following incubation, the sample-contacted surface iswashed to remove non-immunocomplexed material. The washing procedure mayinclude washing with a solution such as PBS/Tween, or a borate buffer.

[0070] Following formation of specific immunocomplexes between the testsample and the bound FlaC protein, analogs, fragments and/or peptides,and subsequent washing, the occurrence, and even amount, ofimmunocomplex formation may be determined by subjecting theimmunocomplex to a second antibody having specificity for the firstantibody. If the test sample is of human origin, the second antibody isan antibody having specificity for human immunoglobulins and in generalIgG. To provide detecting means, the second antibody may have anassociated activity such as an enzymatic activity that will generate,for example, a color development upon incubating with an appropriatechromogenic substrate. Quantification may then achieved by measuring thedegree of color generation using, for example, a visible spectraspectrophotometer.

[0071] 3. Use of Sequences as Hybridization Probes

[0072] The nucleotide sequences of the present invention, comprising thesequence of the FlaC protein, fragments or analogs thereof, now allowfor the identification and cloning of the FlaC protein genes from anyspecies of Campylobacter and other bacteria that have FlaC proteingenes.

[0073] The nucleotide sequences comprising the sequence of the basalbody rod protein genes of the present invention are useful for theirability to selectively form duplex molecules with complementarystretches of other FlaC protein genes. Depending on the application, avariety of hybridization conditions may be employed to achieve varyingdegrees of selectivity of the probe toward the other FlaC protein genes.For a high degree of selectivity, relatively stringent conditions areused to form the duplexes, such as low salt and/or high temperatureconditions, such as provided by 0.02 M to 0.15 M NaCl at temperatures ofbetween about 50° C. to 70° C. For some applications, less stringenthybridization conditions are required such as 0.15 M to 0.9 M salt, attemperatures ranging from between about 20° C. to 55° C. Hybridizationconditions can also be rendered more stringent by the addition ofincreasing amounts of formamide, to destabilize the hybrid duplex. Thus,particular hybridization conditions can be readily manipulated, and willgenerally be a method of choice depending on the desired results. Ingeneral, convenient hybridization temperatures in the presence of 50%formamide are: 42° C. for a probe which is 95 to 100% homologous to thetarget fragment, 37° C. for 90 to 95% homology and 32° C. for 85 to 90%homology.

[0074] In a clinical diagnostic embodiment, the nucleic acid sequencesof the basal body rod protein genes of the present invention may be usedin combination with an appropriate means, such as a label, fordetermining hybridization. A wide variety of appropriate indicator meansare known in the art, including radioactive, enzymatic or other ligands,such as avidin/biotin, which are capable of providing a detectablesignal. In some diagnostic embodiments, an enzyme tag such as urease,alkaline phosphatase or peroxidase, instead of a radioactive tag may beused. In the case of enzyme tags, colorimetric indicator substrates areknown which can be employed to provide a means visible to the human eyeor spectrophotometrically, to identify specific hybridization withsamples containing TfR gene sequences.

[0075] The nucleic acid sequences of FlaC protein genes of the presentinvention are useful as hybridization probes in solution hybridizationsand in embodiments employing solid-phase procedures. In embodimentsinvolving solid-phase procedures, the test DNA (or RNA) from samples,such as clinical samples, including exudates, body fluids or eventissues, is adsorbed or otherwise affixed to a selected matrix orsurface. The fixed, single-stranded nucleic acid is then subjected tospecific hybridization with selected probes comprising the nucleic acidsequences of the FlaC protein genes or fragments thereof of the presentinvention under desired conditions. The selected conditions will dependon the particular circumstances based on the particular criteriarequired depending on, for example, the G+C contents, type of targetnucleic acid, source of nucleic acid, size of hybridization probe etc.Following washing of the hybridization surface so as to removenon-specifically bound probe molecules, specific hybridization isdetected, or even quantified, by means of the label. As with theselection of peptides, it is preferred to select nucleic acid sequenceportions which are conserved among species of bacteria (includingCampylobacter) that produce FlaC proteins. The selected probe may be atleast 18 bp and may be in the range of 30 bp to 90 bp long.

[0076] 4. Expression of the FlaC Flagellin Protein Genes

[0077] Plasmid vectors containing replicon and control sequences whichare derived from species compatible with the host cell may be used forthe expression of the Flac protein genes in expression systems. Thevector ordinarily carries a replication site, as well as markingsequences which are capable of providing phenotypic selection intransformed cells. For example, E. coli may be transformed using pBR322which contains genes for ampicillin and tetracycline resistance and thusprovides easy means for identifying transformed cells. The pBR322plasmid, or other microbial plasmid or phage must also contain, or bemodified to contain, promoters which can be used by the host cell forexpression of its own proteins.

[0078] In addition, phage vectors containing replicon and controlsequences that are compatible with the host can be used as atransforming vector in connection with these hosts. For example, thephage in lambda GEM™-11 may be utilized in making recombinant phagevectors which can be used to transform host cells, such as E. coliLE392.

[0079] Promoters commonly used in recombinant DNA construction includethe b-lactamase (penicillinase) and lactose promoter systems (Refs. 28,29, 30) and other microbial promoters such as the T7 promoter system(U.S. Pat. No. 4,952,496). Details concerning the nucleotide sequencesof promoters are known, enabling a skilled worker to ligate themfunctionally with genes. The particular promoter used will generally bea matter of choice depending upon the desired results. Hosts that areappropriate for expression of the basal body rod protein genes,fragments, analogs or variants thereof include E. coli, Bacillusspecies, Campylobacter, fungi, yeast or the baculovirus expressionsystem may be used.

[0080] In accordance with this invention, it is preferred to make theprotein by recombinant methods, particularly when the naturallyoccurring FlaC protein as purified from a culture of a species ofCampylobacter may include trace amounts of toxic materials or othercontaminants. This problem can be avoided by using recombinantlyproduced FlaC protein in heterologous systems which can be isolated fromthe host in a manner to minimize contaminants in the purified material.Particularly desirable hosts for expression in this regard include Grampositive bacteria which do not have LPS and are therefore endotoxinfree. Such hosts include species of Bacillus and may be particularlyuseful for the production of non-pyrogenic basal body rod proteins,fragments or analogs thereof.

[0081] As noted above, bacteria that lack functional flagella and aresubstantially reduced in motility are also reduced in virulence. Thenucleic acid molecules encoding FlaC proteins of flagella as providedherein allow for the specific modification of flagella (by, for example,site-specific mutagenesis of the genes encoding the basal body proteins)to functionally disable the flagella. Bacteria having such functionallydisabled flagella will be rendered substantially non-motile andsubstantially avirulent. Such avirulent (or attenuated) bacteria areuseful as immunogens for vaccination against disease caused byCampylobacter or other bacteria that produced flagella containing FlaCproteins as encoded by genes of the present invention.

EXAMPLES

[0082] The above disclosure generally describes the present invention. Amore complete understanding can be obtained by reference to thefollowing specific Examples. These Examples are described solely forpurposes of illustration and are not intended to limit the scope of theinvention. Changes in form and substitution of equivalents arecontemplated as circumstances may suggest or render expedient. Althoughspecific terms have been employed herein, such terms are intended in adescriptive sense and not for purposes of limitations.

[0083] Methods of molecular genetics, protein biochemistry, immunologyand fermentation technology used but not explicitly described in thisdisclosure and these Examples are amply reported in the scientificliterature and are well within the ability of those skilled in the art.

Example 1

[0084] This Example describes the analysis of a clone encoding the FlaCprotein of the, flagella of Campylobacter jejuni.

[0085] In ref. 4, are described the cloning and sequencing, of the C.jejuni fur gene and four Fur-box sequences were identified in the 5′flanking region. Oligonucleotides corresponding to the Fur-box sequenceswere used as probes to screen a pBluescript genomic library of C. jejunito attempt to isolate Fur-regulated genes.

[0086] One of the clones, plasmid pD2-2 (FIG. 2) contains the flaC geneof C. jejuni. The flaC gene in plasmid pD2-2 was sequenced by thedideoxy-chain termination method (reference 39 from paper) using theSequenase kit from United States Biomedical or by the cycle sequencingkit from Pharmacia. When necessary, synthetic oligonucleotide primerswere used in order to sequence of both strands. The determined nuclotidesequence (SEQ ID No: 1) is shown in FIG. 1.

[0087] A 16-mer oligonucleotide, 5′ ATTGCGCGAACAGCTG 3′, located on thecomplementary strand of the flaC gene at nt 314 to 298 was used tolocate the transcriptional start point of this gene. Threetranscriptional start points were detected at nt 183, nt 184, and nt185. Using the flaC encoding fragment as a probe, a single hybridizingRNA band of 2.0 kb was seen in a Northern blot analysis.

[0088] The flaC gene (SEQ ID No: 1) is a 747 nt sequence encoding aprotein of 249 amino acids (FIG. 1) (SEQ ID No: 3). The deducedmolecular weight of this protein (FlaC) is 26.6 kDa. Nine nucleotidespreceeding the Met start codon is a good matched Shine-Dalgarnosequence, AGAAGG. At nt 167-174 is the −10 sequence (AATGATTA) and nt145-148 is the −35 sequence (TTAA) of the typical sigma 28 promotersequences (FIG. 1). The stop codon for the flaC gene is located atnucleotide 958.

[0089] Using pulsed field gel electrolysis and the flaC gene as a probein a Southern blot, the gene was mapped to the SalI-D fragment (ref.31). FlgFG genes have been mapped to the same fragment and the flaC geneis located less than 15 kb to the left (counterclockwise) from the basalrod genes (flgFG). The flaC gene is mapped on the opposite side of thegenome from where the flaAB gene cluster is located and are separated byabout 800 to 1000 kb.

[0090] Southern blot hybridization was performed in 30% formamide usinga 0.5 kb PvuII fragment from the coding region of flaC (FIG. 3). Thehybridization identified a single 0.8 kb HindIII band in all the C.jejuni strains tested, a 1.4 kb and a 1.1 kb band were detected in C.coli ATCC 33559 and ATCC 43474, respectively, a 1.8 kb band in C. lariATCC 35221 and PC637 as well as a 0.5 kb band in C. upsaliensis ATCC43954. These results suggest the pressure of a flaC homology among allthe Campylobacter species and strains which were tested.

[0091] Using high stringency hybridization conditions (50% formamide),only the band in the C. jejuni and C. coli strains was detected. For C.jejuni OH4382, the 0.8 kb band was faint, owing to insufficient DNA. Thepresence of the band was confirmed by a subsequent hybridization withthe flaC probe using a filter with equal amounts of genomic DNA from thethree C. jejuni strains. The 0.8 kb band was detected with equalintensity in all three strains.

SUMMARY OF THE DISCLOSURE

[0092] In summary of this disclosure, the flaC gene of Campylobacterjejuni has been cloned and sequenced. The structural organization of thethree flagellin genes in C. jejuni is clearly different as flaC ismapped on the opposite side of the genome from where the flaAB clusterislocated. Expression of the flaC gene can clearly be detected and astrong transcriptional start site can be demonstrated. Modifications arepossible within the scope of this invention.

LIST OF REFERENCES

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[0123] 31. Kim et al, J. Bacteriol. 175: 7468-7470

1 4 1 1105 DNA Campylobacter jejuni 1 atttgttttt tattttacta ataccataattgaactccaa aacttaggcg aaaacactac 60 aaaaactcaa gaatttatca gcatccataatgcaagtgaa gtggtgatta aaatcatctt 120 gataatgcaa gtttttttat atttcttaagctttaaaata gcaaaaaaat gataaaatat 180 taaataaatc caaaagagaa ggagtaggccatgatgatct ctgatgcaac tatgatgcaa 240 caaaattatt atttaaataa tgcacaaaaagctagcgata aagctttaga aaatattgca 300 gctgttcgcg caataagtgg agttgatagtgctaatttag ctattgctga ttctttaaga 360 tctcaatcaa gcactataga tcaaggtgtcgcaaatgctt atgatgctat aggggtttta 420 caaattgcag atgctagcct taccaatatctctcaaagcg cagatagact taatgaactt 480 tcagtaaaaa tgaacaatgc tgcacttaatgattctcaaa aaggaatgct aagaacagaa 540 gcaacacgca tacaagaatc catcaatgattcttttaata atgcaactta taatggaaaa 600 aatgtctttc aaactatgaa ttttgtagtaggtagcggaa ctgaaactac aaatttaaat 660 ccattagcaa cagatggatt aagcatagataatcaagata gtattacaaa ttttatggat 720 caacttggaa gtttaagaag tgaaataggctcaggtatca atgccatcac atcaaatatt 780 aatgcaagtg ttcaaaatag catcaactcaaaagcagctg aaaataattt actaaataat 840 gacatggcaa aaaatgtcaa tgattttaatgccaattatc taaaagaaaa tgctgctgct 900 tttgttgctg cgcaatccaa catgcagcttcaaagcaaaa ttgctaattt attacaataa 960 aataagccct aaatagggct tattttttatcaaaatgact ttagagcaaa ttttagaaaa 1020 aaccaaaaac gttcgtcttg tagcggcaagcaagtatgtc gatgcaagta taattgaaaa 1080 gctttttgat caaggtatag tagaa 1105 2747 DNA Campylobacter jejuni 2 atgatgatct ctgatgcaac tatgatgcaacaaaattatt atttaaataa tgcacaaaaa 60 gctagcgata aagctttaga aaatattgcagctgttcgcg caataagtgg agttgatagt 120 gctaatttag ctattgctga ttctttaagatctcaatcaa gcactataga tcaaggtgtc 180 gcaaatgctt atgatgctat aggggttttacaaattgcag atgctagcct taccaatatc 240 tctcaaagcg cagatagact taatgaactttcagtaaaaa tgaacaatgc tgcacttaat 300 gattctcaaa aaggaatgct aagaacagaagcaacacgca tacaagaatc catcaatgat 360 tcttttaata atgcaactta taatggaaaaaatgtctttc aaactatgaa ttttgtagta 420 ggtagcggaa ctgaaactac aaatttaaatccattagcaa cagatggatt aagcatagat 480 aatcaagata gtattacaaa ttttatggatcaacttggaa gtttaagaag tgaaataggc 540 tcaggtatca atgccatcac atcaaatattaatgcaagtg ttcaaaatag catcaactca 600 aaagcagctg aaaataattt actaaataatgacatggcaa aaaatgtcaa tgattttaat 660 gccaattatc taaaagaaaa tgctgctgcttttgttgctg cgcaatccaa catgcagctt 720 caaagcaaaa ttgctaattt attacaa 747 3249 PRT Campylobacter jejuni 3 Met Met Ile Ser Asp Ala Thr Met Met GlnGln Asn Tyr Tyr Leu Asn 1 5 10 15 Asn Ala Gln Lys Ala Ser Asp Lys AlaLeu Glu Asn Ile Ala Ala Val 20 25 30 Arg Ala Ile Ser Gly Val Asp Ser AlaAsn Leu Ala Ile Ala Asp Ser 35 40 45 Leu Arg Ser Gln Ser Ser Thr Ile AspGln Gly Val Ala Asn Ala Tyr 50 55 60 Asp Ala Ile Gly Val Leu Gln Ile AlaAsp Ala Ser Leu Thr Asn Ile 65 70 75 80 Ser Gln Ser Ala Asp Arg Leu AsnGlu Leu Ser Val Lys Met Asn Asn 85 90 95 Ala Ala Leu Asn Asp Ser Gln LysGly Met Leu Arg Thr Glu Ala Thr 100 105 110 Arg Ile Gln Glu Ser Ile AsnAsp Ser Phe Asn Asn Ala Thr Tyr Asn 115 120 125 Gly Lys Asn Val Phe GlnThr Met Asn Phe Val Val Gly Ser Gly Thr 130 135 140 Glu Thr Thr Asn LeuAsn Pro Leu Ala Thr Asp Gly Leu Ser Ile Asp 145 150 155 160 Asn Gln AspSer Ile Thr Asn Phe Met Asp Gln Leu Gly Ser Leu Arg 165 170 175 Ser GluIle Gly Ser Gly Ile Asn Ala Ile Thr Ser Asn Ile Asn Ala 180 185 190 SerVal Gln Asn Ser Ile Asn Ser Lys Ala Ala Glu Asn Asn Leu Leu 195 200 205Asn Asn Asp Met Ala Lys Asn Val Asn Asp Phe Asn Ala Asn Tyr Leu 210 215220 Lys Glu Asn Ala Ala Ala Phe Val Ala Ala Gln Ser Asn Met Gln Leu 225230 235 240 Gln Ser Lys Ile Ala Asn Leu Leu Gln 245 4 16 DNACampylobacter jejuni 4 attgcgcgaa cagctg 16

What we claim is:
 1. A purified and isolated nucleic acid moleculeencoding a flagellin protein C (FlaC) of a flagellum of a strain ofCampylobacter or a fragment or an analog of the FlaC protein.
 2. Thenucleic acid molecule of claim 1, wherein the strain of Campylobacter isa strain of Campylobacter jejuni.
 3. The nucleic acid molecule of claim1 encoding the FlaC protein of the Campylobacter strain.
 4. A purifiedand isolated nucleic acid molecule having a nucleotide sequence selectedfrom the group consisting of: (a) the entire nucleotide sequence set outin FIG. 1 (SEQ ID No: 1), or the complementary sequence of saidsequence; (b) the coding nucleotide sequence set out in FIG. 1 (SEQ IDNo: 2), or the complementary sequence of said sequence; (c) a nucleotidesequence encoding the amino acid sequence set forth in FIG. 1 (SEQ IDNo: 3); and (d) a nucleotide sequence which hybridizes under stringentconditions to any one of the sequences defined in (a), (b) or (c). 5.The nucleic acid molecule of claim 5, wherein the nucleotide sequencedefined in (b) has at least about 90% sequence identity with any one ofthe sequences defined in (a).
 6. A vector adapted for transformation ofa host comprising the nucleic acid molecule of claim 1 or
 4. 7. Thevector of claim 6 having the identifying characteristics of plasmidpD2-2.
 8. An expression vector adapted for transformation of a hostcomprising the nucleic acid molecule of claim 1 or 4 and expressionmeans operatively coupled to the nucleic acid molecule for expression bythe host of a FlaC protein of a strain of Campylobacter or a fragment oran analog of the FlaC protein.
 9. The expression vector of claim 8,wherein the expression means includes a nucleic acid portion encoding aleader sequence for secretion from the host of the FlaC protein or thefragment or the analog of the FlaC protein.
 10. The expression vector ofclaim 8, wherein the expression means includes a nucleic acid portionencoding a lipidation signal for expression from the host of a lipidatedform of the FlaC protein or the fragment or the analog of the FlaCprotein.
 11. A transformed host containing an expression vector asclaimed in claim
 8. 12. A recombinant FlaC protein or fragment or analogthereof producible by the transformed host of claim
 11. 13. An isolatedand purified FlaC protein of Campylobacter strain substantially freefrom other proteins of the Campylobacter strain.
 14. The FlaC protein ofclaim 13 wherein said strain of Campylobacter is a Campylobacter jejunistrain.
 15. A synthetic peptide having no less than six amino acids andno more than 150 amino acids and containing an amino acid sequencecorresponding to a portion only of a FlaC protein of a strain ofCampylobacter or of an analog of the FlaC protein.
 16. The peptide ofclaim 15, wherein the Campylobacter strain is a Campylobacter jejunistrain.
 17. A live vector for delivery of FlaC protein to a host,comprising a vector containing the nucleic acid molecule of claim 1 or4.
 18. The live vector of claim 17, wherein the vector is selected fromthe group consisting of Salmonella, BCG, adenovirus, poxvirus, vacciniaand poliovirus.
 19. An immunogenic composition, comprising at least oneactive component selected from the group consisting of: (A) a purifiedand isolated nucleic acid molecule encoding a flagellin protein C (FlaC)of a flagellum of a strain of Campylobacter or a fragment or an analogof the FlaC protein; (B) a purified and isolated nucleic acid moleculehaving a sequence selected from the group consisting of: (a) the entirenucleotide sequence set out in FIG. 1 (SEQ ID No: 1), or thecomplementary sequence of said sequence; (b) the coding nucleotidesequence set out in FIG. 1 (SEQ ID No: 2), or the complementary sequenceof said sequence; (c) a nucleotide sequence encoding the amino acidsequence set forth in FIG. 1 (SEQ ID No: 3); and (d) a nucleotidesequence which hybridizes under stringent conditions to any one of thesequences defined in (a), (b) or (c). (C) a recombinant FlaC protein orfragment or analog thereof producible in a transformed host containingan expression vector comprising a nucleic acid molecule as defined in(A) or (B) and expression means operatively coupled to the nucleic acidmolecule for expression by the host of the recombinant FlaC protein orfragment or analog thereof; (D) an isolated and purified FlaC protein ofa Campylobacter strain substantially free from other proteins of theCampylobacter strain; (E) a live vector, comprising a vector containinga purified and isolated nucleic acid molecule encoding a FlaC protein ofa flagellum of a strain of Campylobacter or a fragment or an analog ofthe FlaC protein; (F) a live vector, comprising a vector containing apurified and isolated nucleic acid molecule having a sequence selectedfrom the group consisting of: (a) the entire nucleotide sequence set outin FIG. 1 (SEQ ID No: 1), or the complementary sequence of saidsequence; (b) the coding nucleotide sequence set out in FIG. 1 (SEQ IDNo: 2), or the complementary sequence of said sequence; (c) a nucleotidesequence encoding the amino acid sequence set forth in FIG. 1 (SEQ IDNo: 3); and (d) a nucleotide sequence which hybridizes under stringentconditions to any one of the sequences defined in (a), (b) or (c). (G) asynthetic peptide having no less than six amino acids and no more than150 amino acids and containing an amino acid sequence corresponding to aportion only of a FlaC protein of a strain of Campylobacter or of ananalog of the FlaC protein; and a pharmaceutically acceptable carriertherefor; said at least one active component producing an immuneresponse when administered to a host.
 20. A method for inducingprotection against disease caused by a bacterial pathogen that producesa FlaC protein, comprising administering to a susceptible host aneffective amount of the immunogenic composition of claim
 19. 21. Themethod of claim 20, wherein the bacterial pathogen is a Campylobacterbacterium.
 22. An antiserum or antibody specific for a recombinantprotein as claimed in claim 12, an isolated and purified protein ofclaim 13, a synthetic peptide as claimed in claim 15 or an immunogeniccomposition as claimed in claim
 19. 23. A method of producing a vaccine,comprising: administering the immunogenic composition of claim 19 to atest host to determine an amount and a frequency of administration ofthe active component to confer protection against disease caused by abacterial pathogen that produces the FlaC protein or produces a proteincapable of inducing antibodies in the host specifically reactive withthe FlaC protein, and formulating the active component in a formsuitable for administration to a treated host in accordance with saiddetermined amount and frequency of administration.
 24. The method ofclaim 23 wherein the treated host is a human.
 25. A method ofdetermining the presence of nucleic acid encoding a flagellin protein C(FlaC) of a flagellum of a strain of Campylobacter in a sample,comprising the steps of: (a) contacting the sample with the nucleic acidmolecule of claim 1 or 4 to produce duplexes comprising the nucleic acidmolecule and any said nucleic acid molecule encoding the FlaC protein ofCampylobacter present in the sample and specifically hybridizabletherewith; and (b) determining production of the duplexes.
 26. A methodof determining the presence of a flagellin protein C (FlaC) of aflagellum of a Campylobacter strain in a sample, comprising the stepsof: (a) immunizing a subject with the immunogenic composition of claim19 to produce antibodies specific for said FlaC protein; (b) contactingthe sample with the antibodies to produce complexes comprising any FlaCprotein of a Campylobacter strain present in the sample and said FlaCprotein specific antibodies; and (c) determining production of thecomplexes.
 27. A diagnostic kit for determining the presence of nucleicacid encoding a flagellin protein C (FlaC) protein of a flagellum of astrain of Campylobacter in a sample, comprising: (a) the nucleic acidmolecule of claim 1 or 6; (b) means for contacting the nucleic acid withthe sample to produce duplexes comprising the nucleic acid molecule andany said nucleic acid present in the sample and hybridizable with thenucleic acid molecule; and (c) means for determining production of theduplexes.
 28. A diagnostic kit for detecting the presence of a flagellinprotein C (FlaC) of a flagellum of a Campylobacter strain in a sample,comprising: (a) a FlaC protein specific antibody to the immunogeniccomposition of claim 19; (b) means for contacting the antibody with thesample to produce a complex comprising said FlaC protein and theantibody; and (c) means for determining production of the complex.